This is a project ot study mitochondrial uncoupling protein (UCP) with the long-term goals of elucidating its mechanism and regulation of proton transport and understanding its structural dynamics within the membrane. The specific aims are: 1. To construct and characterize cysteine.less mutants in preparation for studying structural dynamics using site-directed, spin-labeled mutants of uncoupling protein. 2. To identify acid residues in UCP that are critical for anion and proton transport. 4. To test hypotheses relating to the mechanism of UCP-mediated ion transport and its regulation. UCP is a unique protein found exclusively in the brown adipose tissue of mammals. UCP is a primary source of non-shivering thermogenesis, and its physiological role is to provide heat to small mammals and human infants, who are susceptible to cold stress. UCP may also provide a natural, energy-wasting mechanism to mammals. UCP produces heat by dissipating energy from mitochondrial oxidation of fatty acids. It accomplishes this by catalyzing regulated, electrophoretic transport of protons from the cytosol into the mitochondrial matrix. Brown fat and UCP may play a role in the etiology or symptomatology of Sudden Infant Death Syndrome, obesity, alcoholism, and diabetes. This project will use fluorescent probe spectroscopy, electrophysiology, and radioligand binding for functional studies of mutagenized rat UCP. Site-directed mutagenesis will be used to construct variant proteins which are then expressed in a well-established yeast expression system. The expressed protein is purified and reconstituted into lipid bilayer vesicles to study the effects of amino acid substitutions on transport and its regulation. The project includes close collaboration with other laboratories in order to enable study of site-direct, spin-labeled derivatives of UCP by electron paramagnetic resonance spectroscopy.